The endothelial lining of blood vessels provides a barrier between blood and tissues. This permeability barrier is regulated by many factors during both physiological and pathological situations. Recent work has implicated vinculin, a major cytoskeletal protein, in regulating tight junctions. We will explore the role of vinculin in endothelial junctional integrity. We have recently identified a transient interaction between vinculin and the nucleator of actin polymerization, Arp2/3. Our hypothesis is that vinculin is critical in cell junctions because it recruits other components, such as Arp2/3, to these sites. Vinculin mutants that fail to bind specific partners will be expressed in vinculin null cells or endothelial cells in which vinculin levels have been depressed by siRNA techniques. This will allow us to determine which vinculin interactions are needed for junctional integrity. Many agents diminish vascular permeability by elevating endothelial cAMP levels. We will test the hypothesis that elevated cAMP exerts this effect by phosphorylation of RhoA on serine188, which, in turn, promotes its interaction with RhoGDI, extraction from the membrane and sequestration. We have generated RhoA mutants that cannot be phosphorylated that should block this pathway, and have developed an antibody that detects phosphorylated RhoA that we will use to monitor its phosphorylation state under different conditions. RhoGDI acts as a cytosolic reservoir for Rho family proteins, but little is known about how they are released to distinct membrane compartments. We hypothesize that there are proteins that bind to the C-terminus of RhoA promoting release from RhoGDI and targeting to specific membranes. We will use affinity isolation techniques to look for such proteins. One factor that increases vascular permeability is VEGF. It has been shown previously to activate Rac, and in preliminary work, we have found that it activates Rho. Our final aim will test the hypothesis that VEGF activation of Rho or Rac regulates junctional integrity and endothelial permeability. We will attempt to identify guanine nucleotide exchange factors activated by VEGF, using nucleotide-free Rho proteins that bind exchange factors with high affinity. The role of exchange factors downstream from VEGF will be tested by expressing dominant negative versions of these proteins or by inhibiting their expression with siRNA.